Supports for immobilizing DNA or the like

ABSTRACT

A subject of the present invention is to provide a support with immobilized DNA as a DNA library and particularly to a support suitable for reproducing DNA in accordance with a DNA amplification reaction. In the present invention, DNA or the like is immobilized on a surface of a support made of one kind or more than materialSelected from a group of a diamond including non-diamond carbon, amorphous carbon and graphite. A hydroxyl group may be bonded to a surface of the support or a group bonded to a hydrocarbon group and a carboxyl group at its terminal end may be bonded to a surface of the support through an ester linkage or a peptide linkage.

REFERENCE TO RELATED APPLICATIONS

The present application is the national stage under 35 U.S.C. §371 ofinternational application PCT/JP99/05712, filed Oct. 15, 1999, whichdesignated the United States, and which application was not published inthe English language.

INDUSTRIAL FIELD

The present invention relates to supports for immobilizing DNA or thelike, particularly to supports which are chemically modified, moreparticularly to supports which are chemically modified by a hydroxylgroup, a carboxyl group and so on at the terminal thereof.

BACKGROUND OF THE INVENTION

In a conventional process, in order to obtain a specific amount oftarget DNA, the following heat cycle 1) to 3) has to be repeated in aDNA amplification reaction and so on:

1) the temperature of a test material is increased to 95° C. in order tobreak the hydrogen bonds of the double chain;

2) the temperature of the test material is decreased to 45° C. in orderto reproduce DNA by adding a primer; and

3) the temperature of the test material is increased to 74° C. in orderto reproduce DNA by extending the primer with heat-resistant polymerase.

In such a DNA amplificaiton reaction, the test material is filled into acontainer made from synthetic resin. The container is installed in analuminum block and heat-cycled.

However, it requires many hours to accomplish the heat cycle. Itrequires several hours to obtain a target amount of DNA. There is adrawback that others kinds of DNA are also reproduced in addition to thetarget DNA since the accuracy of thermal control is low.

To resolve the above drawback, a subject of the present invention is toprovide a support suitable for immobilizing DNA easily and reproducingDNA by a DNA amplification reaction.

DISCLOSURE OF THE INVENTION

A support according to the present invention is comprises at least onecomponent selected from the group of diamond, including non-diamondcarbon, amorphous carbon, and graphite.

The support preferably has a chemical modification such as polar group,hydroxyl group or carboxyl group at its terminal.

The carboxyl group is preferably linked to a surface of the supportthrough an ester linkage or a peptide linkage.

BEST MODE FOR CARRYING OUT THE INVENTION

Regarding carbon material such as diamond including non-diamond carbon,amorphous carbon and graphite and so on, carbons are exposed on asurface of the support so that the surface can be chemically modifiedwith a hydroxyl group, a carboxyl group and so on and DNA and so on canbe immobilized easily. It is the most suitable to reproduce DNA and soon by a DNA amplification reaction.

Even if a surface of the support according to the present invention iscontaminated, chemical modification can be reproduced by hydrolyzing.

The support according to the present invention comprises diamondincluding non-diamond carbon, amorphous carbon, graphite and so on.Although any methods for producing the surface can be used, a microwaveplasma CVD method, an ECR CVD method, a high frequency plasma CVDmethod, an IPC method, a DC sputtering method, an ECR sputtering method,an ion plating method, an arc ion plating method, an EB evaporationmethod, a resistance heating evaporation method and so on arepreferable. For example, carbon may be amorphous carbon with hydrogenobtained by steam-baking a resist layer made of polyimide material.Further, carbon may be sintered slurrying resin mixed with graphitepowder. In the present invention, carbon may be selected from one ormore than materials described above.

A surface of the support according to the present invention may beroughened. Such a rough surface has a relatively large surface area onwhich a large amount of DNA can be immobilized. The shape of the supportmay be a plate shape, a ball shape, polygon shape or various shapes. Thematerial of the support may be mixed with the above described materialand the other components. The support according to the present inventionmay be exposed on a surface of an object.

In the next, a specific group is chemically modified (added) on asurface of the above support. By providing a chemical modification, DNAis able to be immobilized on the surface of the support. A hydroxylgroup, a carboxyl group, a surface group, a cyano group, a nitro group,a thiol group, an amino group and so on may be utilized as a specificgroup having a polar group and chemically modified on a surface of thesupport. In addition, an organic carbonic acid is also available.

The carboxyl group may be bonded to a terminal of the support throughthe other hydrocarbon group. In such a case, it is preferable that thenumber of carbons in the hydrocarbon group is from zero to 10 in orderto immobilize DNA. Regarding the acid which can be charged to be ahydrocarbon group, a mono-carboxylic acid such as formic acid, aceticacid, propionic acid, di-carboxylic acid such as oxalic acid, malonicacid, succinic acid, maleinic acid, fumaric acid, phthalic acid and apolycarboxylic acid such as trimellitic acid are considerable.

In the case of utilizing the support according to the present inventionfor a DNA amplification reaction, there are two options, that is, oneoption is to require a hydrolysis-resist characteristic and anotheroption is to require a reproduction of chemical modification byhydrolyzing.

In the case of requiring the hydrolysis-resist characteristic, it ispreferable that a carboxyl group bonded to a terminal end of thehydro-carbon group is bonded to a surface of the support through apeptide linkage in order to provide alkali-proof characteristic.

On the other hand, in the case of requiring a reproduction of chemicalmodification by hydrolyzing and removing the produced chemicalmodification, it is preferable that a carboxyl group bonded to aterminal end of the hydro-carbon group is bonded to a surface of thesupport through an ester linkage.

Regarding a method for bonding a hydroxyl group to a terminal end of ahydro-carbon group on the surface of the support, a method for oxidizinga surface of the support with oxygen plasma and treating with steam, amethod for chloridizing a surface of the support by irradiating ultraviolet beam in chloride gas and hydrolyzing the support by hydrolyzingin alkali solution are suitable.

Methods for linking the carboxyl group bonded to the terminal end of thehydro-carbon group on the surface of the support through the peptidelinkage, include chloridizing a surface of the support by irradiatingwith ultra violet radiation in chloride gas, aminatizing the support byirradiating with ultra violet radiation in ammonia gas, reacting withcarbonic chloride in a non-water soluble solution and neutralizing inalkaline solution.

Methods for bonding the carboxyl group connected to a terminal end ofthe hydrocarbon group on a surface of the support through an esterlinkage, include chloridizing the surface of the support by irradiatingwith ultra violet radiation in chloride gas, reacting with sodiumcarbonate in non-water soluble solution and neutralizing in a weak acidsolution and a oxidizing a surface of the support with oxygen plasma,chloridizing, hydrolyzing by hydrolyzing in alkali solution, reactingwith carbonic chloride in non-water soluble solution and neutralizing inalkaline solution.

EXAMPLES

Examples according to the present invention will be described in detailhereinafter.

Example 1

A diamond circular plate including non-diamond carbon of which adiameter is 64 mm, a thickness is 0.1 mm and a surface roughness Ra is0.5 mm is vapor-phased by a microwave plasma CVD method. Regarding apeak ratio of non-diamond carbon (uncompleted diamond) with respect todiamond carbon (completed diamond), an area of 10 mm×10 mm of thecircular plate is analyzed by Raman spectroscopic analysis method sothat an existence of non-diamond carbon can be recognized. A test pieceincluding the non-diamond carbon of 10 mm×10 mm is cut off from thecircular plate.

In the example 1, after the surface of the test piece is oxidized withoxygen plasma energized by microwave, the test piece is set in aseparable flask. After instituting atmosphere in the flask with steam,the atmosphere is heated at 400° C. for 30 minutes while steam is addedto the flask. After the test piece is cooled, the test piece is pickedup and dried so as to obtain a support having a hydroxyl group at itsterminal end.

In accordance with a SIMS method, the test piece is cut off and treatedwith oxygen plasma and steam in order. Then, each peak strength ofhydrogen and hydroxyl group is measured, respectively. In the case ofthe peak strength of hydrogen as 1, the peak strength ratio of thehydroxyl group is as follows.

Peak strength ratio of hydroxyl group After cutting off test piece 0.11After treating with oxygen plasma 0.61 After treating with steam 1.09

As shown in the above table, the peak strength ratio of the hydroxylgroup is increased by processing the oxygen plasma a treatment, thesteam treatment. Therefore, it can be recognized that a surface of thesupport is chemically modified by a hydroxyl group.

Example 2

A diamond circular plate including non-diamond carbon of which adiameter is 64 mm, a thickness is 0.1 mm and a surface roughness Ra is0.3 mm is vapor-phased by a microwave plasma CVD method. An area of 10mm×10 mm of the circular plate is analyzed by Raman spectroscopicanalysis method. As the result, all the carbon are non-diamond carbon(uncomplicated diamond). A test piece of 10 mm×10 mm is cut off from thecircular plate by laser beam.

The test piece is set in a separable flask. After flushing the flaskwith argon gas, the test piece is irradiated by ultra violet beamsproduced by a Hg—Xe lamp of which a main wavelength is 3600 Å for 60minutes in order to chloridize a surface of the test piece whilechloride gas is introduced into the flask per 1SCCM. After flushing theatmosphere with argon gas, the test piece is picked up. The test pieceis boiled is sodium hydroxide solution of 10 wt % for 15 minutes. Afterwashing with water and drying, a support having a hydroxyl group at aterminal end is obtained.

In accordance with the SIMS method, each peak strength of hydrogen, thehydroxyl group and the chloride group is measured at a timing beforechloridizing, a timing after chloridizing and a timing after treatingwith sodium hydroxide in order. In the case of the peak strength ofhydrogen as 1, the peak strength ratio of the hydroxyl group and thechloride group are as follows.

Peak strength ratio Hydroxyl group Chloride group Before chloridizing0.11 — After chloridizing 0.17 0.47 After treating with 0.45 0.31 sodiumhydroxide

As described above, the peak strength ratio of the hydroxyl group isgradually increased by conducting the chloridizing treatment and thesodium hydroxide treatment. Thus, it is recognized that a surface of thesupport is chemically modified with the hydroxyl group. Judging fromdecreasing the ratio of the chloride group, it is recognized that thechloride group is instituted to the hydroxyl group.

Example 3

A diamond circular plate including complete diamond carbon andnon-diamond carbon having a diameter of 64 mm, a thickness of 0.1 mm anda surface roughness Ra of 0.5 mm is vapor-phased by a microwave plasmaCVD method. Regarding a peak ratio of non-diamond carbon (uncompleteddiamond) with respect to diamond carbon (completed diamond), an area of10 mm×10 mm of the circular plate is analyzed by the Raman spectroscopicanalysis method so that an existence of non-diamond carbon can berecognized. A test piece including the non-diamond carbon of 10 mm×10 mmis cut off from the circular plate. After oxidizing a surface of thetest piece with oxygen plasma energized by microwave, a surface of thetest piece is chloridized. After flushing atmosphere with argon gas, thetest piece is picked up. The test piece is boiled in sodium hydroxidesolution of 10 wt % for 15 minutes. After being cleaned with water anddried, a support having a hydroxyl group at its terminal end isobtained.

In accordance with the SIMS method, after polishing a surface of thetest piece, each peak strength of hydrogen, the hydroxyl group and thechloride group is measured at a timing before oxygen plasma treatment, atiming after oxygen plasma treatment, a timing after chloridizing and atiming after sodium hydroxide treatment in order.

Peak strength ratio Hydroxyl group Chloride group Before oxygen 0.11 —plasma treatment After oxygen 0.67 — plasma treatment Chloridizing 0.190.44 sodium hydroxide 0.50 0.30 treatment

As shown in the above Table, the peak strength ratio of the hydroxylgroup is gradually increased by passing the oxygen plasma treatment,chloridizing and sodium hydroxide so that it can be recognized a surfaceof the support is chemically modified with the hydroxyl groups. Judgingfrom decreasing the chloride group, it is recognized that the chlorideis instituted with the hydroxyl group.

Example 4

A graphite circular plate is formed by a sputtering method. A test pieceof 10 mm×10 mm is cut off from the circular plate by laser beam. Thetest piece is set in a separable flask. After flushing the atmosphere inthe flask with argon gas, the test piece is irradiated by ultra violetbeams produced by a Hg—Xe lamp of which a main wavelength is 3600 Å for60 minutes in order to chloridize a surface of the test piece whilechloride gas is introduced into the flask per 1SCCM. After adding argongas to the atmosphere in the flask again, N-dimethyl formamide solutionof 100 ml of sodium salt of sebacic acid of 1 wt % is added. A condenseris provided at the separable flask and refluxed for two hours. Then, thetest piece is picked up. After cleaning the test piece with acetic acidsolution of 1 wt %, the test piece is further cleaned with acetone anddried. Thus is obtained a support having a carboxyl group linked withthe sebacic acid through an ester linkage at a terminal.

In accordance with the SIMS method, each peak strength of hydrogen,hydroxyl group and the chloride group is measured at a timing beforechloridizing, a timing after chloridizing and a timing after sebacicsoda treatment in order. In the case of the peak strength of hydrogen as1, the peak strength ratio of the hydroxyl group and the chloride groupare as follows.

Peak strength ratio Hydroxyl group Chloride group Before chloridizing0.11 — After chloridizing 0.17 0.47 Sebacic soda 0.35 0.31 treatment

As shown in the above Table, the peak strength ratio of the hydroxylgroup is gradually increased by passing the chloridizing treatment andthe successive sodium salt of sebacic acid treatment. In accordance withthe FTIR method, absorption strength caused by stretching vibrationbetween carbon and hydrogen on a surface of the test piece andabsorption strength caused by stretching vibration between carbon andoxygen are measured. The absorption strength of the both cases areincreased (an increasing ratio of the absorption strength with respectto a surface blank of the test piece is about 30%). Judging from thefact, it is recognized that the surface of the support is chemicallymodified by a group bonded to the carboxyl group at its terminal end ofhydrocarbon group of the sebacic acid. The chloride group is decreasedso that it can be recognized the chloride group is instituted with thehydroxyl group.

Example 5

A resist layer made from polyimide system material is baked with steamso as to form a thin plate made from amorphous carbon includinghydrogen. A test piece of 10 mm×10 mm is cut off from the thin plate bylaser beam. After oxidizing a surface of the test piece with oxygenplasma energized by microwave, a surface of the test piece ischloridized. After flushing the atmosphere with argon gas, the testpiece is picked up. The test piece is boiled in potassium hydroxidesolution of 10 wt % for 15 minutes so as to modify the surface of thetest piece with hydroxyl group. After drying the test piece, the testpiece is set in a separable flask with a condenser with a calciumchloride dry tube provided at an upper portion of the separable flask.Chloroform of 50 ml and triethylamine of 1 g are added and theatmosphere in the separable flask is flushed with argon gas. While theseparable flask is cooled with ice, chloroform solution of 50 ml inwhich 10 g of succinyl chloride is dissolved is gradually added. Afterrefluxing for four hours, the test piece is picked up. The test piece iscleaned with potassium carbonate solution of 10 wt % and then cleanedwith acetone. After drying the test piece, there is obtained a supporthaving a carboxyl group bonded to malonic acid through an ester linkageat its terminal end.

In accordance with a SIMS method, each peak strength of hydrogen andhydroxyl group is measured at a timing before oxygen plasma treatment, atiming after oxygen plasma treatment, a timing after chloridizing, atiming after hydrolyzing and a timing after succinyl chloride treatmentin order. In the case of the peak strength of hydrogen as 1, the peakstrength ratio of the hydroxyl group is as follows.

Peak strength ratio of hydroxyl group Before oxygen plasma 0.11treatment After oxygen plasma 0.66 treatment After chloridizing 0.19After hydrolyzing 0.65 After succinyl chloride 0.46 treatment

As shown in the above Table, the peak strength ratio of the hydroxylgroup is gradually increased by passing the oxygen plasma treatment, thechloridizing treatment, the hydrolyzing treatment and the succinylchloride treatment. In accordance with the FTIR method, absorptionstrength caused by stretching vibration between carbon and hydrogen on asurface of the test piece and absorption strength caused by stretchingvibration between carbon and oxygen are measured. The absorptionstrength of the both cases are increased (an increasing ratio of theabsorption strength with respect to a surface blank of the test piece isabout 25%). Judging from the fact, it is recognized that a surface ofthe support is chemically modified by a group bonded to the carboxylgroup at its terminal end of hydrocarbon group of the malonic acid.

Example 6

A graphite circular plate having a diameter of 64 mm, a thickness of 0.1mm and a surface roughness Ra of 0.3 mm is vapor-phased by vaporizingslurry type solvent mixed with graphite powder and resin powder. In thecircular plate, an area of 10 mm×10 mm of the circular plate is analyzedby Raman spectroscopic analysis method so that all of the diamond carbonare non-diamond carbon (amorphous carbon). A test piece of 10 mm×10 mmis cut off from the circular plate. After hydrogenating a surface of thetest piece by hydrogen plasma energized by microwave, the surface of thetest piece is chloridized. After setting the test piece in a separableflask, the atmosphere in the separable flask is flushed with argon gas.While ammonia gas is introduced to the flask per 1SCCM, the surface ofthe test piece is aminated by irradiating ultra violet of which a mainwavelength is 3600 Å produced by a Hg—Xe lamp for 60 minutes. Afterflushing the atmosphere with argon gas, a condenser with a chloridecalcium dry tube is provided at an upper portion of the separable flask.Chloroform of 50 ml is added and the atmosphere is flushed with argongas.

While the separable flask is cooled with ice, chloroform solution of 50ml mixed with succinyl chloride of 10 g is gradually added. Afterrefluxing for 4 hours, the test piece is picked up and cleaned withpotassium carbonate solution of 10 wt %. The test piece is furthercleaned with acetone and dried so that a support having the carboxylgroup bonded to malonic acid through a peptide linkage at its terminalend can be obtained.

In accordance with the SIMS method, each peak strength of hydrogen,hydroxyl group and chloride group is measured at a timing beforechloridizing, a timing after chloridizing, a timing of succinyl chloridetreatment in order. In the case of the peak strength of hydrogen as 1,the peak strength of the hydroxyl group and the chloride group are shownas follows.

Peak strength ratio Hydroxyl group Chloride group Before chloridizing0.11 — After chloridizing 0.19 0.45 After aminating 0.16 0.10 Aftersuccinyl 0.55 0.10 chloride treatment

As shown in the above Table, the peak strength ratio of the hydroxylgroup increases step by step by passing the hydrogen plasma treatment,the successive chloridizing treatment, the hydrolyzing treatment and thesuccinyl chloride treatment. In accordance with the FTIR method,absorption strength caused by stretching vibration between carbon andhydrogen on a surface of the test piece and absorption strength causedby stretching vibration between carbon and oxygen are measured. Theabsorption strength of the both cases are increased (an increasing ratioof the absorption strength with respect to a surface blank of the testpiece is about 25%).

Judging from the fact, it is recognized that a surface of the support ischemically modified by a group bonded to the carboxyl group at aterminal end of hydroxide group of the malonic acid.

A DNA amplification reaction is operated on supports chemically modifiedwith the carboxyl group at the terminal end as described in the examples1 to 6, and a target amount of DNA can be obtained within 1 hour.

By utilizing a support chemically modified according to the presentinvention, a terminal end of the oligonucleotide is immobilized to ahydroxyl group or a carboxyl group a its terminal through a hydrogenlinkage. Further DNA having complementary base sequence with respect tothe oligonucleotide is immobilized so as to use as a DNA library chip.Instead of DNA, nucleotide, oligonucleotide, DNA fragment and so on canbe immobilized on a surface of a diamond support as a library.

POSSIBILITY OF USE IN THE INVENTION

A support according to the present invention comprises carbon materialso that DNA can be immobilized easily on the support and a DNAamplification reaction is operated very easily.

In the support according to the present invention, a surface of thesupport is chemically modified with a hydroxyl group or a carboxyl groupand so on so that an immobilization of DNA and so on can become stableand the support can be utilized as a chip for reproducing DNA by the DNAamplification reaction.

Even if a surface of the support according to the present invention iscontaminated, the chemical modification can be reproduced by hydrolysis.A production cost of a DNA chip can be saved.

What is claimed is:
 1. A support for immobilizing DNA comprising atleast one material selected from the group consisting of diamond,uncompleted diamond, and amorphous carbon, wherein said material isproduced by microwave plasma CVD, ECR CVD, high frequency plasma CVD,IPC, DC sputtering, ECR sputtering, ion plating, arc ion plating, EBevaporation, or resistance heating evaporation, said support beingchemically modified by a polar group bonded to the surface of thesupport such that DNA is immobilized thereon.
 2. The support accordingto claim 1 wherein a hydroxyl group, a sulfate group, a cyano group, anitro group, a thiol group, an amino group, or a carboxyl group at thesurface of the support is bonded to the support.
 3. The supportaccording to claim 2 wherein the support is chemically modified by acarboxyl group, and the carboxyl group is bonded to a surface of saidsupport through an ester linkage containing a hydrocarbon chain havingfrom 1 to 10 carbon atoms.
 4. The support according to claim 2 whereinthe support is chemically modified by a carboxyl group and the carboxylgroup is bonded to the surface of the support through a peptide linkage.5. The support according to claim 1 wherein the DNA is immobilized onthe support through a hydrogen linkage at a terminal end of a hydroxylgroup on the support or through a carboxyl group of an oligonucleotide.6. The support according to claim 1 and wherein a hydroxal group isbonded to the surface of the support for immobilizing DNA thereon. 7.The support according to claim 2 wherein a hydroxyl group is bonded tothe surface of the support.